Nanoparticle immunoconjugates

ABSTRACT

Disclosed herein are nanoparticle immunoconjugates useful for therapeutics and/or diagnostics. The immunoconjugates have diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution). In certain embodiments, the conjugates are silica-based nanoparticles with single chain antibody fragments attached thereto.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Application Ser. No.62/144,278 filed on Apr. 7, 2015 and U.S. Application Ser. No.62/151,943 filed on Apr. 23, 2015, the disclosures of which are herebyincorporated by reference in their entireties.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. U54CA199081-01 awarded by NIH. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates generally to nanoparticle immunoconjugates (e.g.,under 20 nanometers in diameter), useful, for example, for thedetection, prevention, and/or treatment of cancer and other diseases.

BACKGROUND

Nano-therapeutic and/or -diagnostic delivery vehicles are typicallymacro- or supra-molecular multicomponent systems, ranging in size from1-1,000 nm, that are either inherently therapeutic (e.g., no activepharmaceutical ingredient) or function as therapeutic or diagnosticdelivery systems. To date, liposomal nanoparticles and biologicscomprise a large proportion of the number of FDA-approved products orproducts in clinical trials used to treat and/or detect a variety ofcancer types, while a number of polymer-based particle formulations arecurrently in early phase trials.

Desirable candidates for nanotherapeutic delivery systems share a commonfeature of incorporating and releasing a drug compound in a controlledmanner, which can favorably alter drug bioavailability andpharmacokinetics, while minimizing off-target toxicities. Ideally, animaging label is incorporated therein to assess their preciselocalization and retention at disease sites.

However, these systems function using different mechanisms. For example,antibody drug conjugates (ADCs) achieve lower drug toxicity primarilythrough active targeting of tumor cells and conditional release of drugmolecules. Upon binding a cell surface antigen, active drug releaseoccurs after cellular internalization and endosomal uptake. On the otherhand, liposomes and polymer-based drug delivery systems, which aretypically much larger assembled complexes (˜20-150 nm diameters)passively loaded with a greater payload (˜10,000 drug molecules forDoxil) or imaging agents, have generally lacked targeting capabilities(BIND-014 is an exception). Therefore, these complexes rely primarily onthe well-known enhanced permeability and retention (EPR) effect for thesuccessful delivery of nano-formulated drugs. While interstitialpermeation of liposomes may be poor due to their size, the free drug isreleased through various mechanisms that are not entirely understood.For example, Abraxane (˜140 nm) relies on a different approach toenhance the bioavailability of a hydrophobic compound. In this case, aspecific formulation of albumin and drug (paclitaxel) forms the initialcomplex, which is in turn estimated to disperse into smallerprotein-drug aggregates upon injection.

Metastatic disease may effectively be treated with immunotherapies;however, a significant subpopulation will not respond due to lack ofantigenic mutations or the immune-evasive properties of cancer. Inaddition, although radiation therapy (RT) is a standard treatment forcancer, local failures occur. Preclinical data indicate that RT canpotentiate the systemic efficacy of immunotherapy, while activation ofthe innate and adaptive immune system can enhance the local efficacy ofRT.

There remains a need for a platform that can be used for the detection,prevention, and/or treatment of cancer and other diseases.

SUMMARY

Described herein are target-specific nanoparticle immunoconjugates(e.g., single chain antibody fragments bound to the particle surface)for targeted diagnostic and/or therapeutic platforms. In certainembodiments, the nanoparticle immunoconjugates are less than 20 nm(e.g., 6 to 10 nm) in diameter. This small size is found to offeradvantages in therapeutic and/or imaging applications. For example, thedisclosed immunoconjugates may offer improved targeting of diseasedtissue and reduced non-specific uptake by organs (e.g., by the liver).The smaller immunoconjugates may also demonstrate reduced immunereactivity, thereby further improving efficacy.

Also described herein is a multi-therapeutic platform that comprises animmunoconjugate and therapeutic radioisotopes. In certain embodiments,immunoconjugates and therapeutic radioisotopes are delivered in concertfor synergistic effects of combined radiation therapy and immunotherapy.In certain embodiments, an antibody fragment and a therapeuticradioisotope are attached to nanoparticles, thereby creating atarget-specific nanoparticle immunoconjugate. A given nanoparticle canhave both radionuclides (radioisotopes) and antibodies (and/or antibodyfragments) attached thereto (in which case, the immunoconjugate is aradioimmunoconjugate). Also, in some embodiments, a portion of theadministered nanoparticles have radionuclides attached (covalently ornon-covalently bonded, or otherwise associated with the nanoparticle)while other administered nanoparticles have antibody fragments attached.Also included in various embodiments are combination therapies in whicheither exiting (e.g., traditional) radiotherapy is combined withadministration of nanoparticle immunoconjugates described herein, orexisting (e.g., traditional) immunotherapy is combined withadministration of nanoparticle radioconjugates (nanoparticles with boundradioisotopes),

The certain embodiments, the target-specific nanoparticleimmunoconjugates comprise a targeting peptide. In certain embodiments,the therapeutic radioisotope is delivered separately from thetarget-specific nanoparticle immunoconjugate (e.g., via radiationtherapy or via attached to a separate target-specific nanoparticle). Incertain embodiments, immunotherapy is delivered separately from thetarget-specific immunoconjugate. In certain embodiments, an antibodyfragment is attached to one polyethylene glycol (PEG) moiety (via aparticular chelator) and a radioisotope is attached to another PEGmoiety (via another chelator). The PEG moieties are then attached tonanoparticles.

In one aspect, the invention is directed to An immunoconjugatecomprising: a nanoparticle; and an antibody fragment conjugated to thenanoparticle, wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm).

In certain embodiments, the antibody fragment is covalently ornon-covalently bonded to the nanoparticle via a linker or covalently ornon-covalently bonded directly to the nanoparticle, or associated withthe nanoparticle or a composition surrounding the nanoparticle, e.g.,via van der Waals forces.

In certain embodiments, the nanoparticle is coated with an organicpolymer (e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugatecomprises a chelator).

In certain embodiments, a targeting peptide (e.g., alphaMSH, any peptideknown to be immunomodulatory and anti-inflammatory in nature).

In certain embodiments, the antibody fragment is in a range from about 5kDa to about 25 kDa (e.g., from about 10 kDa to about 20 kDa, e.g.,about 15 kDa) (e.g., wherein the antibody fragment comprises afunctional single domain antibody fragment).

In certain embodiments, the antibody fragment is from about 20 kDa toabout 45 kDa (e.g., from about 25 kDa to about 30 kDa) (e.g., whereinthe antibody fragment comprises a functional single chain antibodyfragment).

In certain embodiments, the antibody fragment is from about 40 kDa toabout 80 kDa (e.g., from about 50 kDa to about 70 kDa, e.g., about 60kDa) (e.g., wherein the antibody fragment comprises a functional fabfragment).

In certain embodiments, the nanoparticle comprises silica.

In certain embodiments, the nanoparticle comprises a silica-based coreand a silica shell surrounding at least a portion of the core.

In certain embodiments, the nanoparticle comprises a fluorescentcompound within the core.

In certain embodiments, the antibody fragment is a member selected fromthe set consisting of a recombinant antibody fragment (fAbs), a singlechain variable fragment (scFv), and a single domain antibody (sdAb)fragment.

In certain embodiments, the antibody fragment is a single chain variablefragment (scFv).

In certain embodiments, the antibody fragment is a single domain (sdAb)fragment.

In certain embodiments, the nanoparticle (a single nanoparticle) hasfrom one to ten antibody fragments (e.g., from 1 to 7, e.g., from 1 to5, e.g., from 2 to 7, e.g., from 2 to 5, e.g., from 1 to 4, e.g., from 2to 4) attached thereto.

In certain embodiments, the antibody fragment is conjugated to thenanoparticle via a PEG moiety and a chelator.

In certain embodiments, the nanoparticle has a diameter (e.g., averagediameter) no greater than 15 nanometers (e.g., no greater than 13nanometers, e.g., no greater than 10 nanometers).

In certain embodiments, the nanoparticle has a diameter (e.g., averagediameter) in a range from 1 nm to 20 nm (e.g., from 2 nm to 15 nm, e.g.,from 5 nm to 15 nm, e.g., from 1 nm to 10 nm, e.g., from 2 nm to 10 nm,e.g., from 5 nm to 10 nm).

In certain embodiments, the antibody fragment comprises a memberselected from the set consisting of anti-CEA scFv, anti-GPIIb/IIIa,anti-VEGF-A, and anti-TNF-α (e.g., PEGylated).

In certain embodiments, the immunoconjugate comprises one or moreimaging agents (e.g., within the nanoparticle, attached to thenanoparticle, and/or attached to the antibody fragment).

In certain embodiments, the one or more imaging agents comprise a PETtracer (e.g., ⁸⁹Zr, ⁶⁴Cu, and/or [¹⁸F] fluorodeoxyglucose).

In certain embodiments, the one or more imaging agents comprise afluorophore (e.g., a cyanine).

In certain embodiments, the immunoconjugate further comprises atherapeutic agent (e.g., wherein the therapeutic agent is attached tothe nanoparticle, or to the antibody fragment, or to both thenanoparticle and the antibody fragment, e.g., wherein the attachment iscovalent or non-covalent).

In certain embodiments, the therapeutic agent comprises a chemotherapydrug (e.g., sorafenib, paclitaxel, docetaxel, MEK162, etoposide,lapatinib, nilotinib, crizotinib, fulvestrant, vemurafenib, bexorotene,and/or camptotecin).

In certain embodiments, the therapeutic agent comprises a radioisotope(e.g., wherein the radioisotope is attached to the nanoparticle via asecond chelator) (e.g., wherein the radioisotope is a therapeuticradioisotope).

In certain embodiments, the radioisotope is a member selected from thegroup consisting of ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁷⁷Lu, ⁶⁷Cu, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N, ¹⁵O, ¹⁸F, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹³Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au,¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ⁸⁹Zr, ²²⁵Ac, and¹⁹²Ir.

In another aspect, the invention is directed to a method of treating adisease or condition, the method comprising administering to a subject apharmaceutical composition comprising the immunoconjugate (e.g., totarget a particular type of tissue, e.g., cancer).

In certain embodiments, the method comprises administering a therapeuticradioisotope (e.g., wherein the therapeutic radioisotope is attached toa second nanoparticle having a diameter (e.g., average diameter) nogreater than 20 nanometers (e.g., as measured by dynamic lightscattering (DLS) in aqueous solution, e.g., saline solution) (e.g.,wherein the radioisotope is attached to the second nanoparticle via asecond chelator)) (e.g., wherein the second nanoparticle has a diameterfrom 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g.,from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm).

In another aspect, the invention is directed to a method of treating adisease or condition, the method comprising administering to a subject apharmaceutical composition comprising the immunoconjugate (e.g., totarget a particular type of tissue, e.g., cancer) (e.g., for combinedradiation therapy and immunotherapy).

In certain embodiments, the pharmaceutical composition further comprisesa carrier.

In another aspect, the invention is directed to a method of in vivoimaging (e.g., intraoperative imaging), the method comprising:administering to a subject a composition comprising the immunoconjugate(e.g., such that the immunoconjugate preferentially collects in aparticular region, e.g., near or within a particular tissue type, e.g.,cancer), wherein the immunoconjugate comprises an imaging agent; anddetecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging agent.

In another aspect, the invention is directed to a method of making theimmunoconjugate, the method comprising: contacting ananoparticle-PEG-thiol with a protein-maleimide, thereby producing theimmunoconjugate.

In certain embodiments, the method further comprises reacting thenanoparticle with one or more compounds, the one or more compoundscomprising a thiol moiety and an amine moiety (e.g., cysteine methylester or cysteamine-HCl), thereby producing a nanoparticle-PEG-amine;reacting the nanoparticle-PEG-amine with SPDP, then removing a pyridine2-thione from the product (e.g., using TCEP), thereby producing thenanoparticle-PEG-thiol.

In another aspect, the invention is directed to a method of making theimmunoconjugate, the method comprising: modifying the antibody fragment(protein) with a first click reactive group (e.g.,methyltetrazine-PEG4-NHS ester; modifying a nanoparticle-PEG-amine witha click partner of the first click reactive group (e.g., TCO-PEG4-NHSester); and reacting the modified antibody fragment with the modifiednanoparticle-PEG, thereby producing the immunoconjugate.

In certain embodiments, the method further comprises reacting thenanoparticle with one or more compounds, the one or more compoundscomprising a thiol moiety and an amine moiety (e.g., cysteine methylester or cysteamine-HCl), thereby producing the nanoparticle-PEG-amine.

In another aspect, the invention is directed to a method of treating adisease or condition, the method comprising administering to a subject acomposition (e.g., a pharmaceutical composition) comprising: ananoparticle; and a therapeutic radioisotope conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., to target aparticular type of tissue, e.g., cancer).

In certain embodiments, the method comprises administering immunotherapy(e.g., wherein the immunotherapy comprises administering to a subject apharmaceutical composition comprising the immunoconjugate).

In another aspect, the invention is directed to an immunoconjugatecomprising: a nanoparticle; and an antibody fragment conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g.,wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator), for use in a method of treating a disease or condition in asubject, wherein the treating comprises: delivering the immunoconjugateto the subject; and delivering a therapeutic radioisotope (e.g., whereinthe therapeutic radioisotope is attached to a second nanoparticle havinga diameter (e.g., average diameter) no greater than 20 nanometers (e.g.,as measured by dynamic light scattering (DLS) in aqueous solution, e.g.,saline solution) (e.g., wherein the radioisotope is attached to thesecond nanoparticle via a second chelator)).

In another aspect, the invention is directed to an immunoconjugatecomprising: a nanoparticle; a therapeutic radioisotope (e.g., whereinthe radioisotope is attached to the nanoparticle via a second chelator)(e.g., wherein the radioisotope is a therapeutic radioisotope); and anantibody fragment conjugated to the nanoparticle (e.g., covalently ornon-covalently bonded to the nanoparticle via a linker or covalently ornon-covalently bonded directly to the nanoparticle, or associated withthe nanoparticle or a composition surrounding the nanoparticle, e.g.,via van der Waals forces), wherein the nanoparticle has a diameter(e.g., average diameter) no greater than 20 nanometers (e.g., asmeasured by dynamic light scattering (DLS) in aqueous solution, e.g.,saline solution) (e.g., wherein the average nanoparticle diameter isfrom 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g.,from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g.,wherein the immunoconjugate has an average diameter no greater than 50nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., nogreater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than10 nm) (e.g., wherein the nanoparticle is coated with an organic polymer(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugatecomprises a chelator) for use in a method of treating a disease orcondition in a subject, wherein the treating comprises: delivering theimmunoconjugate to the subject.

In another aspect, the invention is directed to an immunoconjugatecomprising a nanoparticle; and an antibody fragment conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g.,wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator), and wherein the immunoconjugate comprises an imaging agent,for use in a method of in vivo diagnosis of a disease or condition in asubject, wherein the in vivo diagnosis comprises: delivering theimmunoconjugate to the subject; and detecting (e.g., via PET, X-ray,MRI, CT, etc.) the imaging agent.

In another aspect, the invention is directed to an immunoconjugatecomprising: a nanoparticle; and an antibody fragment conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g.,wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator), and wherein the immunoconjugate comprises an imaging agent,for use in (a) a method of treating a disease or condition in a subjector (b) a method of in vivo diagnosis of a disease or condition in asubject, wherein the method comprises: administering to a subject apharmaceutical composition comprising the immunoconjugate (e.g., totarget a particular type of tissue, e.g., cancer); and optionally,detecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging agent.

In another aspect, the invention is directed to an immunoconjugatecomprising a nanoparticle; and an antibody fragment conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g.,wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator) for use in therapy.

In another aspect, the invention is directed to an immunoconjugatecomprising: a nanoparticle; a therapeutic radioisotope (e.g., whereinthe radioisotope is attached to the nanoparticle via a second chelator)(e.g., wherein the radioisotope is a therapeutic radioisotope); and anantibody fragment conjugated to the nanoparticle (e.g., covalently ornon-covalently bonded to the nanoparticle via a linker or covalently ornon-covalently bonded directly to the nanoparticle, or associated withthe nanoparticle or a composition surrounding the nanoparticle, e.g.,via van der Waals forces), wherein the nanoparticle has a diameter(e.g., average diameter) no greater than 20 nanometers (e.g., asmeasured by dynamic light scattering (DLS) in aqueous solution, e.g.,saline solution) (e.g., wherein the average nanoparticle diameter isfrom 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g.,from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g.,wherein the immunoconjugate has an average diameter no greater than 50nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., nogreater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than10 nm) (e.g., wherein the nanoparticle is coated with an organic polymer(e.g., polyethylene glycol (PEG)) (e.g., wherein immunoconjugatecomprises a chelator) for use in therapy.

In another aspect, the invention is directed to an immunoconjugatecomprising: a nanoparticle; and an antibody fragment conjugated to thenanoparticle (e.g., covalently or non-covalently bonded to thenanoparticle via a linker or covalently or non-covalently bondeddirectly to the nanoparticle, or associated with the nanoparticle or acomposition surrounding the nanoparticle, e.g., via van der Waalsforces), wherein the nanoparticle has a diameter (e.g., averagediameter) no greater than 20 nanometers (e.g., as measured by dynamiclight scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., wherein the average nanoparticle diameter is from 1 to 20 nm,e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm,e.g., from 4 to 10 nm, e.g., from 4 to 8 nm) (e.g., wherein theimmunoconjugate has an average diameter no greater than 50 nm, e.g., nogreater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g.,wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator), and wherein the immunoconjugate comprises an imaging agent,for use in in vivo diagnosis.

In another aspect, the invention is directed to a composition (e.g.,pharmaceutical composition) comprising: a nanoparticle; and atherapeutic radioisotope conjugated to the nanoparticle (e.g.,covalently or non-covalently bonded to the nanoparticle via a linker orcovalently or non-covalently bonded directly to the nanoparticle, orassociated with the nanoparticle or a composition surrounding thenanoparticle, e.g., via van der Waals forces), wherein the nanoparticlehas a diameter (e.g., average diameter) no greater than 20 nanometers(e.g., as measured by dynamic light scattering (DLS) in aqueoussolution, e.g., saline solution) (e.g., wherein the average nanoparticlediameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm)(e.g., wherein the nanoparticle is coated with an organic polymer (e.g.,polyethylene glycol (PEG)) (e.g., wherein immunoconjugate comprises achelator)) for use in a method of treating a disease or condition in asubject, wherein the treating comprises: delivering the composition tothe subject; and delivering immunotherapy (e.g., wherein theimmunotherapy comprises administering to a subject a pharmaceuticalcomposition comprising the immunoconjugate).

Elements of embodiments involving one aspect of the invention (e.g.,methods) can be applied in embodiments involving other aspects of theinvention (e.g., systems), and vice versa.

Definitions

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration”: The term “administration” refers to introducing asubstance into a subject. In general, any route of administration may beutilized including, for example, parenteral (e.g., intravenous), oral,topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal,rectal, nasal, introduction into the cerebrospinal fluid, orinstillation into body compartments. In certain embodiments,administration is oral. Additionally or alternatively, in certainembodiments, administration is parenteral. In certain embodiments,administration is intravenous.

“Antibody”: As used herein, the term “antibody” refers to a polypeptidethat includes canonical immunoglobulin sequence elements sufficient toconfer specific binding to a particular target antigen. Intactantibodies as produced in nature are approximately 150 kD tetramericagents comprised of two identical heavy chain polypeptides (about 50 kDeach) and two identical light chain polypeptides (about 25 kD each) thatassociate with each other into what is commonly referred to as a“Y-shaped” structure. Each heavy chain is comprised of at least fourdomains (each about 110 amino acids long)—an amino-terminal variable(VH) domain (located at the tips of the Y structure), followed by threeconstant domains: CH₁, CH₂, and the carboxy-terminal CH₃ (located at thebase of the Y's stem). A short region, known as the “switch”, connectsthe heavy chain variable and constant regions. The “hinge” connects CH₂and CH₃ domains to the rest of the antibody. Two disulfide bonds in thishinge region connect the two heavy chain polypeptides to one another inan intact antibody. Each light chain is comprised of two domains—anamino-terminal variable (VL) domain, followed by a carboxy-terminalconstant (CL) domain, separated from one another by another “switch”.Intact antibody tetramers are comprised of two heavy chain-light chaindimers in which the heavy and light chains are linked to one another bya single disulfide bond; two other disulfide bonds connect the heavychain hinge regions to one another, so that the dimers are connected toone another and the tetramer is formed. Naturally-produced antibodiesare also glycosylated, typically on the CH₂ domain. Each domain in anatural antibody has a structure characterized by an “immunoglobulinfold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets)packed against each other in a compressed antiparallel beta barrel. Eachvariable domain contains three hypervariable loops known as “complementdetermining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant“framework” regions (FR1, FR2, FR3, and FR4). When natural antibodiesfold, the FR regions form the beta sheets that provide the structuralframework for the domains, and the CDR loop regions from both the heavyand light chains are brought together in three-dimensional space so thatthey create a single hypervariable antigen binding site located at thetip of the Y structure. The Fc region of naturally-occurring antibodiesbinds to elements of the complement system, and also to receptors oneffector cells, including for example effector cells that mediatecytotoxicity. Affinity and/or other binding attributes of Fc regions forFc receptors can be modulated through glycosylation or othermodification. In certain embodiments, antibodies produced and/orutilized in accordance with the present invention include glycosylatedFc domains, including Fc domains with modified or engineered suchglycosylation. For purposes of the present invention, in certainembodiments, any polypeptide or complex of polypeptides that includessufficient immunoglobulin domain sequences as found in naturalantibodies can be referred to and/or used as an “antibody”, whether suchpolypeptide is naturally produced (e.g., generated by an organismreacting to an antigen), or produced by recombinant engineering,chemical synthesis, or other artificial system or methodology. Incertain embodiments, an antibody is polyclonal; in certain embodiments,an antibody is monoclonal. In certain embodiments, an antibody hasconstant region sequences that are characteristic of mouse, rabbit,primate, or human antibodies. In certain embodiments, antibody sequenceelements are humanized, primatized, chimeric, etc, as is known in theart. Moreover, the term “antibody” as used herein, can refer inappropriate embodiments (unless otherwise stated or clear from context)to any of the art-known or developed constructs or formats for utilizingantibody structural and functional features in alternative presentation.For example, embodiments, an antibody utilized in accordance with thepresent invention is in a format selected from, but not limited to,intact IgG, IgE and IgM, bi- or multi-specific antibodies (e.g.,Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs,cameloid antibodies, masked antibodies (e.g., Probodies®), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain or Tandem diabodies(TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrinrepeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody,Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®,MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®. In certainembodiments, an antibody may lack a covalent modification (e.g.,attachment of a glycan) that it would have if produced naturally. Incertain embodiments, an antibody may contain a covalent modification(e.g., attachment of a glycan, a payload [e.g., a detectable moiety, atherapeutic moiety, a catalytic moiety, etc], or other pendant group[e.g., poly-ethylene glycol, etc.]).

“Antibody fragment”: As used herein, an “antibody fragment” includes aportion of an intact antibody, such as, for example, the antigen-bindingor variable region of an antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)2, and Fv fragments; triabodies; tetrabodies;linear antibodies; single-chain antibody molecules; and multi specificantibodies formed from antibody fragments. For example, antibodyfragments include isolated fragments, “Fv” fragments, consisting of thevariable regions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy chain variable regionsare connected by a peptide linker (“ScFv proteins”), and minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region. In many embodiments, an antibody fragment containssufficient sequence of the parent antibody of which it is a fragmentthat it binds to the same antigen as does the parent antibody; incertain embodiments, a fragment binds to the antigen with a comparableaffinity to that of the parent antibody and/or competes with the parentantibody for binding to the antigen. Examples of antigen bindingfragments of an antibody include, but are not limited to, Fab fragment,Fab′ fragment, F(ab′)2 fragment, scFv fragment, Fv fragment, dsFvdiabody, dAb fragment, Fd′ fragment, Fd fragment, and an isolatedcomplementarity determining region (CDR) region. An antigen bindingfragment of an antibody may be produced by any means. For example, anantigen binding fragment of an antibody may be enzymatically orchemically produced by fragmentation of an intact antibody and/or it maybe recombinantly produced from a gene encoding the partial antibodysequence. Alternatively or additionally, antigen binding fragment of anantibody may be wholly or partially synthetically produced. An antigenbinding fragment of an antibody may optionally comprise a single chainantibody fragment. Alternatively or additionally, an antigen bindingfragment of an antibody may comprise multiple chains which are linkedtogether, for example, by disulfide linkages. An antigen bindingfragment of an antibody may optionally comprise a multimolecularcomplex. A functional single domain antibody fragment is in a range fromabout 5 kDa to about 25 kDa, e.g., from about 10 kDa to about 20 kDa,e.g., about 15 kDa; a functional single-chain fragment is from about 10kDa to about 50 kDa, e.g., from about 20 kDa to about 45 kDa, e.g., fromabout 25 kDa to about 30 kDa; and a functional fab fragment is fromabout 40 kDa to about 80 kDa, e.g., from about 50 kDa to about 70 kDa,e.g., about 60 kDa.

“Associated”: As used herein, the term “associated” typically refers totwo or more entities in physical proximity with one another, eitherdirectly or indirectly (e.g., via one or more additional entities thatserve as a linking agent), to form a structure that is sufficientlystable so that the entities remain in physical proximity under relevantconditions, e.g., physiological conditions. In certain embodiments,associated moieties are covalently linked to one another. In certainembodiments, associated entities are non-covalently linked. In certainembodiments, associated entities are linked to one another by specificnon-covalent interactions (e.g., by interactions between interactingligands that discriminate between their interaction partner and otherentities present in the context of use, such as, for examplestreptavidin/avidin interactions, antibody/antigen interactions, etc.).Alternatively or additionally, a sufficient number of weakernon-covalent interactions can provide sufficient stability for moietiesto remain associated. Exemplary non-covalent interactions include, butare not limited to, electrostatic interactions, hydrogen bonding,affinity, metal coordination, physical adsorption, host-guestinteractions, hydrophobic interactions, pi stacking interactions, vander Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, etc.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe materials that do not elicit a substantial detrimental responsein vivo. In certain embodiments, the materials are “biocompatible” ifthey are not toxic to cells. In certain embodiments, materials are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and/or their administration in vivo does notinduce inflammation or other such adverse effects. In certainembodiments, materials are biodegradable.

“Biodegradable”: As used herein, “biodegradable” materials are thosethat, when introduced into cells, are broken down by cellular machinery(e.g., enzymatic degradation) or by hydrolysis into components thatcells can either reuse or dispose of without significant toxic effectson the cells. In certain embodiments, components generated by breakdownof a biodegradable material do not induce inflammation and/or otheradverse effects in vivo. In certain embodiments, biodegradable materialsare enzymatically broken down. Alternatively or additionally, in certainembodiments, biodegradable materials are broken down by hydrolysis. Incertain embodiments, biodegradable polymeric materials break down intotheir component polymers. In certain embodiments, breakdown ofbiodegradable materials (including, for example, biodegradable polymericmaterials) includes hydrolysis of ester bonds. In certain embodiments,breakdown of materials (including, for example, biodegradable polymericmaterials) includes cleavage of urethane linkages.

“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution saline solutions and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

“Imaging agent”: As used herein, “imaging agent” refers to any element,molecule, functional group, compound, fragments thereof or moiety thatfacilitates detection of an agent (e.g., a polysaccharide nanoparticle)to which it is joined. Examples of imaging agents include, but are notlimited to: various ligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F,³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ¹³¹I, ⁶⁴Cu, ⁶⁸Ga, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y,^(99m)Tc, ¹⁷⁷Lu, ⁸⁹Zr etc.), fluorescent dyes (for specific exemplaryfluorescent dyes, see below), chemiluminescent agents (such as, forexample, acridinum esters, stabilized dioxetanes, and the like),bioluminescent agents, spectrally resolvable inorganic fluorescentsemiconductors nanocrystals (i.e., quantum dots), metal nanoparticles(e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagneticmetal ions, enzymes (for specific examples of enzymes, see below),colorimetric labels (such as, for example, dyes, colloidal gold, and thelike), biotin, dioxigenin, haptens, and proteins for which antisera ormonoclonal antibodies are available. The radionuclides may be attachedvia click chemistry, for example.

“Protein”: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least 3-5 amino acids linked to one another bypeptide bonds). Proteins may include moieties other than amino acids(e.g., may be glycoproteins, proteoglycans, etc.) and/or may beotherwise processed or modified. In certain embodiments “protein” can bea complete polypeptide as produced by and/or active in a cell (with orwithout a signal sequence); in certain embodiments, a “protein” is orcomprises a characteristic portion such as a polypeptide as produced byand/or active in a cell. In certain embodiments, a protein includes morethan one polypeptide chain. For example, polypeptide chains may belinked by one or more disulfide bonds or associated by other means. Incertain embodiments, proteins or polypeptides as described herein maycontain L-amino acids, D-amino acids, or both, and/or may contain any ofa variety of amino acid modifications or analogs known in the art.Useful modifications include, e.g., terminal acetylation, amidation,methylation, etc. In certain embodiments, proteins or polypeptides maycomprise natural amino acids, non-natural amino acids, synthetic aminoacids, and/or combinations thereof. In certain embodiments, proteins areor comprise antibodies, antibody polypeptides, antibody fragments,biologically active portions thereof, and/or characteristic portionsthereof.

“Pharmaceutical composition”: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In certain embodiments,active agent is present in unit dose amount appropriate foradministration in a therapeutic regimen that shows a statisticallysignificant probability of achieving a predetermined therapeutic effectwhen administered to a relevant population. In certain embodiments,pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to theskin, lungs, or oral cavity; intravaginally or intrarectally, forexample, as a pessary, cream, or foam; sublingually; ocularly;transdermally; or nasally, pulmonary, and to other mucosal surfaces.

“Substantially”: As used herein, the term “substantially”, and grammaticequivalents, refer to the qualitative condition of exhibiting total ornear-total extent or degree of a characteristic or property of interest.One of ordinary skill in the art will understand that biological andchemical phenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result.

“Subject”: As used herein, the term “subject” includes humans andmammals (e.g., mice, rats, pigs, cats, dogs, and horses). In manyembodiments, subjects are be mammals, particularly primates, especiallyhumans. In certain embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; poultry such as chickens,ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. In certain embodiments (e.g.,particularly in research contexts) subject mammals will be, for example,rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine suchas inbred pigs and the like.

“Therapeutic agent”: As used herein, the phrase “therapeutic agent”refers to any agent that has a therapeutic effect and/or elicits adesired biological and/or pharmacological effect, when administered to asubject.

“Therapeutically effective amount”: as used herein, is meant an amountthat produces the desired effect for which it is administered. Incertain embodiments, the term refers to an amount that is sufficient,when administered to a population suffering from or susceptible to adisease, disorder, and/or condition in accordance with a therapeuticdosing regimen, to treat the disease, disorder, and/or condition. Incertain embodiments, a therapeutically effective amount is one thatreduces the incidence and/or severity of, and/or delays onset of, one ormore symptoms of the disease, disorder, and/or condition. Those ofordinary skill in the art will appreciate that the term “therapeuticallyeffective amount” does not in fact require successful treatment beachieved in a particular individual. Rather, a therapeutically effectiveamount may be that amount that provides a particular desiredpharmacological response in a significant number of subjects whenadministered to patients in need of such treatment. In certainembodiments, reference to a therapeutically effective amount may be areference to an amount as measured in one or more specific tissues(e.g., a tissue affected by the disease, disorder or condition) orfluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those ofordinary skill in the art will appreciate that, in certain embodiments,a therapeutically effective amount of a particular agent or therapy maybe formulated and/or administered in a single dose. In certainembodiments, a therapeutically effective agent may be formulated and/oradministered in a plurality of doses, for example, as part of a dosingregimen.

“Treatment”: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In certain embodiments, treatmentmay be of a subject who has been diagnosed as suffering from therelevant disease, disorder, and/or condition. In certain embodiments,treatment may be of a subject known to have one or more susceptibilityfactors that are statistically correlated with increased risk ofdevelopment of the relevant disease, disorder, and/or condition.

Drawings are presented herein for illustration purposes, not forlimitation.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe present disclosure will become more apparent and better understoodby referring to the following description taken in conduction with theaccompanying drawings, in which:

FIG. 1 shows a schematic illustration showing the synthesis of⁸⁹Zr-labeled C′dot radioimmunoconjugate using a chelator-basedradiolabeling technique. PEGylated and maleimide-functionalized C′ dot(C′ dot-PEG-Mal, 1) was first reacted with reduced glutathione (GSH) tointroduce the —NH₂ groups for the following-up bioconjugates, forming C′dot-PEG-GSH (2). Then the nanoparticle was conjugated with DBCO-PEG4-NHSester and DFO-NCS, forming C′ dot-PEG-DBCO (3) and DFO-C′ dot-PEG-DBCO(4), respectively. Azide-functionalized small targeting ligands, such assingle-chain variable fragment (scFv-azide) (or single-domain antibody,sdAb-azide), was conjugated to the nanoparticle based on strain-promotedazide-alkyne cycloaddition, forming DFO-C′ dot-PEG-scFv (5). The finalC′dot radioimmunoconjugate (⁸⁹Zr-DFO-C′ dot-PEG-scFv, 6) was by labelingit with ⁸⁹Zr-oxalate. The embodiments illustrated in FIG. 1 are notlimited to scFv and can include various types of antibody fragments,e.g., sdAbs.

FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B) sagittalPET images of ⁸⁹Zr-DFO-C′ dot-PEG at different post-injection timepoints (10 min, 1 h, Day 1, Day 3 and Day 6) in a healthy nude mouse.The reaction ratio between C′ dot-PEG-Mal and GSH was kept at 1:20. ThePET images were acquired by using a Focus 120 MicroPET scanner.

FIG. 3 shows biodistribution data of ⁸⁹Zr-DFO-C′ dot-PEG in a healthynude mouse on Day 6. Less than 2% ID/g of bone (and joint) uptake wasobserved.

FIGS. 4A and 4B show a chelator-free ⁸⁹Zr radiolabeling experimentalexample.

FIG. 4A shows ⁸⁹Zr labeling yields of C′ dot-PEG-Mal under varied pHconditions at 75° C.

FIG. 4B shows ⁸⁹Zr labeling yields of C′ dot-PEG-Mal using variedcombinations of C′ dot to ⁸⁹Zr-oxalate ratio.

FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C′ dot-PEG atdifferent post-injection time points (10 min, Day 1, Day 3 and Day 6) ina healthy nude mouse. [⁸⁹Zr]C′ dot-PEG was synthesized by using achelator-free radiolabeling technique. The PET images were acquired byusing a Focus 120 MicroPET scanner.

FIG. 5A shows PET images acquired without EDTA(ethylenediaminetetraacetic acid).

FIG. 5B shows PET images acquired with EDTA

FIG. 6 shows biodistribution data of [⁸⁹Zr]C′ dot-PEG in healthy nudemice (n=3) on Day 7. Over 10% ID/g of bone (and joint) uptake wasobserved in this case, indicating a less stable radiolabeling using achelator-free method (when compared with that of chelator-based method).

FIG. 7 shows biodistribution data of ⁸⁹Zr-DFO-C′ dot, ⁸⁹Zr-DFO-C′dot-DBCO and ⁸⁹Zr-DFO-C′ dot-PEG-sdAb in healthy nude mice at 48 hpost-injection. An improved pharmacokinetic profile (with prolongedblood circulation half-life and lower liver uptake) can be achieved byoptimizing the number of DFO, DBCO and sdAb from each C′ dot.

FIG. 8 shows an exemplary schematic of thiol-maleimide chemistry.

FIG. 9 shows an exemplary schematic of alkene-tetrazine chemistry.

DETAILED DESCRIPTION

Throughout the description, where compositions are described as having,including, or comprising specific components, or where methods aredescribed as having, including, or comprising specific steps, it iscontemplated that, additionally, there are compositions of the presentinvention that consist essentially of, or consist of, the recitedcomponents, and that there are methods according to the presentinvention that consist essentially of, or consist of, the recitedprocessing steps.

It should be understood that the order of steps or order for performingcertain action is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

Molecular therapeutics (e.g., antibodies) can modulate the immune systemtoward antitumor activity by manipulating immune checkpoints (e.g., themonoclonal antibody ipilimumab inhibits CTLA4, a negative regulatorymolecule that inhibits function of the immune system). The rationale isto trigger preexisting, but dormant, antitumor immune responses. Othermolecules and pathways have acted as immune switches. PD-1, anothernegative regulatory receptor expressed on T cells, has also beentargeted. Switching a single immune checkpoint may not be sufficient toinduce an antitumor response, explaining some of the failures oftargeting single immune regulatory checkpoints like PD-1 or CTLA4.However, without wishing to be bound to any theory, treatment can bebolstered by the addition of RT, which is thought, in some cases, tohave immunomodulatory properties. In these cases, tumors outside of RTtreatment fields have been found to shrink as a result of a putativesystemic inflammatory or immune response provoked by RT, highlightingthe potential for radiation to spark a systemic antitumor immuneresponse. Augmenting immune activity may also potentiate the localeffects of RT.

By raising the concentration alone of these immunoconjugates, diseasecan be treated. A therapeutic radiolabel can also be added to furthertreat disease. In certain embodiments, the immunoconjugate act as atherapeutic at high concentrations, and without a therapeuticradiolabel. In certain embodiments, the radiolabel is attached to thesame nanoparticle in an all-in-one multi-therapeutic platform.Alternatively, therapeutic radioisotopes can be administeredindependently.

Described herein are target-specific nanoparticle immunoconjugates(e.g., single chain antibody fragments bound to the particle surface)for targeted diagnostic and/or therapeutic platforms. In certainembodiments, the nanoparticle immunoconjugates are less than 20 nm(e.g., 6 to 10 nm) in diameter. This small size is found to offeradvantages in therapeutic and/or imaging applications. For example, thedisclosed immunoconjugates may offer improved targeting of diseasedtissue and reduced non-specific uptake by organs (e.g., by the liver).The smaller immunoconjugates may also demonstrate reduced immunereactivity, thereby further improving efficacy.

In certain embodiments, the nanoparticle comprises silica, polymer(e.g., poly(lactic-co-glycolic acid) (PLGA)), and/or metal (e.g., gold,iron).

In certain embodiments, the silica-based nanoparticle platform comprisesultrasmall nanoparticles or “C dots,” which are fluorescent,organo-silica core shell particles that have diameters controllable downto the sub-10 nm range with a range of modular functionalities. C dotsare described by U.S. Pat. No. 8,298,677 B2 “Fluorescent silica-basednanoparticles”, U.S. Publication No. 2013/0039848 A1 “Fluorescentsilica-based nanoparticles”, and U.S. Publication No. US 2014/0248210 A1“Multimodal silica-based nanoparticles”, the contents of which areincorporated herein by reference in their entireties. Incorporated intothe silica matrix of the core are near-infrared dye molecules, such asCy5.5, which provides its distinct optical properties. Surrounding thecore is a layer or shell of silica. The silica surface is covalentlymodified with silyl-polyethylene glycol (PEG) groups to enhancestability in aqueous and biologically relevant conditions. Theseparticles have been evaluated in vivo and exhibit excellent clearanceproperties owing largely to their size and inert surface. Among theadditional functionalities incorporated into C dots are chemicalsensing, non-optical (PET) image contrast and in vitro/in vivo targetingcapabilities, which enable their use in visualizing lymph nodes forsurgical applications, and melanoma detection in cancer.

C dots are synthesized via an alcohol-based modified Stöber process.C′dots are synthesized in water.

C dots or C′dots provide a unique platform for drug delivery due totheir physical properties as well as demonstrated human in vivocharacteristics. These particles are ultrasmall and benefit from EPReffects in tumor microenvironments, while retaining desired clearanceand pharmacokinetic properties. To this end, described herein is ananoparticle drug delivery system in which, in certain embodiments, drugconstructs are covalently attached to C dots or C′dots (or othernanoparticles).

C dots or C′dots can serve as highly specific and potentmulti-therapeutic targeted particle probes to combine antibody fragmentswith therapeutic radiolabels (e.g., ¹⁷⁷Lu, ²²⁵Ac, ⁹⁰Y, ⁸⁹Zr) on a singleplatform. Alternatively, C dot or C′dot coupling of targeting peptides,such as alphaMSH, known to be immunomodulatory and anti-inflammatory innature, can also be combined with C dot or C′dot radiotherapeutic(and/or other particle-based) platforms to achieve enhanced efficacy. Incertain embodiments, the concentration of the radioisotope and/orantibody fragment is higher in therapeutic applications compared todiagnostic applications.

In contrast to other multimodal platforms, immunoconjugates can comprisedifferent moieties that are attached to the nanoparticle itself. Forexample, in certain embodiments, a radioisotope is attached to thenanoparticle and an antibody fragment is attached to thenanoparticle—that is, in these embodiments, the radiolabel is notattached to the antibody fragment itself. As another example,immunoconjugates can comprise a targeting ligand attached to thenanoparticle, a radioisotope attached to the nanoparticle, and anantibody fragment attached to the nanoparticle. The stoichiometricratios of different moieties attached to the C dot will affect thebiodistribution of the nanoparticle immunoconjugate.

The immunoconjugates, e.g., C dot-antibody (mAb) and -antibody-fragment(vFab) conjugates, can be prepared using either of two approaches.Scheme 1 comprises thiol-maleimide chemistry, as shown in FIG. 8 .Scheme 1 is designed around proteins modified to contain maleimidegroups. Scheme 2 comprises alkene-tetrazine chemistry as shown in FIG. 9.

In Scheme 1 as shown in FIG. 8 , C dots containing Cy5 dye, surfacefunctionalized with PEG and maleimide groups (C dots-(Cy5)-PEG-mal) wereprepared as previously described in Bradbury et al., 2014. Silanesmodified with the Cy5 fluorophore were prepared and titrated withtetramethylorthosilane (TMOS) into a dilute solution of NH₄OH (molarratio TMOS:Cy5:NH3:H20 is 1:0.001:0.44:1215) and allowed to mix for 24hours (Urata C, Aoyama Y, Tonegawa A, Yamauchi Y, Kuroda K. Dialysisprocess for the removal of surfactants to form colloidal mesoporoussilica nanoparticles. Chem Commun (Camb). 2009; (34):5094-6) (Yamada H,Urata C, Aoyama Y, Osada S, Yamauchi Y, Kuroda K. Preparation ofColloidal Mesoporous Silica Nanoparticles with Different Diameters andTheir Unique Degradation Behavior in Static Aqueous Systems, Chem.Mater. 2012; 24(8):1462-71.) (Wang J, Sugawara-Narutaki A, Fukao M,Yokoi T, Shimojima A, Okubo T. Two-phase synthesis of monodispersesilica nanospheres with amines or ammonia catalyst and their controlledself-assembly. ACS Appl Mater Interfaces. 2011; 3(5):1538-44.) Thisresulted in a Cy5 encapsulated silica particle, the surface of which wasfurther PEGylated and functionalized with maleimide groups by treatmentwith PEG-silane (500 g/mole) (Suzuki K, Ikari K, Imai H. Synthesis ofsilica nanoparticles having a well-ordered mesostructured using a doublesurfactant system. J Am Chem Soc. 2004; 126(2):462-3) andmaleimide-PEG-silane (molar ratio PEG-silane:TMOS:mal-PEG-silane of1:2.3:0.006). The maleimide groups can then be effectively transformedinto amine groups by reacting the particles with compounds that containa thiol and amine (e.g., cysteine methyl ester or cysteamine-HCl). Theresulting C dot-(Cy5)-PEG-amine can then be subsequently modified with asuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). The pyridyldithiolserves at least two purposes: one, it can be used to quantitateconjugation efficiencies; two, it may serves as a ‘protecting group’ tominimize oxidation of thiol groups; etc. TCEP can then be used to removethe group releasing a pyridine 2-thione, which can be measured by HPLCor UV-absorption for quantitation. The resulting C dot-(Cy5)-PEG-thiolcan then be reacted with protein-maleimide leading to the desired Cdot-(Cy5)-PEG-mAb or C dot-(Cy5)-PEG-vFab.

In Scheme 2 as shown in FIG. 9 , alkene-tetrazine chemistry is utilizedfor protein attachment. Here, the mAb or vFab is modified with a clickreactive groups, such as methyltetrazine-PEG₄-NHS ester. The Cdot-(Cy5)-PEG-amine, as described in FIG. 8 (Scheme 1), is then modifiedwith the appropriate click partner, (e.g., TCO-PEG4-NHS ester). In thefinal step, the methyltetrazine-mAb or -vFab can then be reacted withthe C dot-(Cy5)-PEG-TCO leading to the C dot-(Cy5)-PEG-mAb or Cdot-(Cy5)-PEG-vFab product.

Antibody fragments (fAbs) provide advantages (e.g., size, no Fc regionfor reduced immunogenicity, scalability, and adaptability) compared tostandard monoclonal antibodies (mAbs). fAbs are the stripped-downbinding region of an antibody which is usually expressed as a singlecontinuous sequence in an expression host (e.g., E. Coli). In certainembodiments, a fAb or mAb can be as small as 15 kDa (+/−5 kDa) (e.g.,about 3 nm). In other embodiments, a fAb or mAb can be up to 150 kDa(e.g., up to 20 nm). In one embodiment, a fAb is approximately 60 kDa(e.g., +/−15 kDa). A fAb comprises an immunoglobin heavy-chain variableand constant domain linked to the corresponding domains of animmunoglobin light chain. In another embodiment, the antibody format canbe a single chain variable fragment (scFv) fragment that isapproximately 30 kDa (e.g., +/−10 kDa). A scFv fragment comprises aheavy-chain variable domain linked to a light-chain variable domain. Inother embodiments, the antibody format can be a single domain antibody(sdAb) fragment that is approximately 15 kDa (e.g., +/−5 kDa). A sdAbfragment comprises a single heavy-chain variable domain. In certainembodiments, the antibody fragment is an anti-CEA scFv for targetingdifferent tumors.

In certain embodiments, various linkers are used. In certainembodiments, a cleavable linker (e.g., peptide, hydrazine, or disulfide)is used. In certain embodiments, a noncleavable linker (e.g., thioether)is used. In certain embodiments, a peptide linker is selectively cleavedby lysosomal proteases (e.g., cathepsin-B). In certain embodiments, avaline-citrulline dipeptide linker is used.

In certain embodiments, different linkers as described in U.S. Pat. Nos.4,680,338, 5,122,368, 5,141,648, 5,208,020, 5,416,064, 5,475,092,5,543,390, 5,563,250 5,585,499, 5,880,270, 6,214,345, 6,436,931,6,372,738, 6,340,701, 6,989,452, 7,129,261, 7,375,078, 7,498,302,7,507,420, 7,691,962, 7,910,594, 7,968,586, 7,989,434, 7,994,135,7,999,083, 8,153,768, 8,236,319, Zhao, R.; et al, (2011) J. Med. Chem.36, 5404; Doronina, S.; et al, (2006) Bioconjug Chem, 17, 114; Hamann,P.; et al. (2005) Bioconjug Chem. 16, 346, the contents of which arehereby incorporated by reference herein, are used.

In certain embodiments, the mAbs and/or fAbs are U.S. approved forcertain uses. Non-limiting examples of mAbs and fAbs includeanti-GPIIb/IIIa, anti-VEGF-A, and anti-TNF-α. ReoPro (abciximab) is ananti-GPIIb/IIIa, chimeric fAb, IgG1-κ developed by Centocor/Eli Lilly asdescribed by Nelson and Reichert, “Development trends for therapeuticantibody fragments,” Nature Biotechnology, 27(4), 2009. Lucentis(ranibizumab) is an anti-VEGF-A, humanized Fab IgG1-κ developed byGenentech (Nelson and Reichert, 2009) that is used to prevent wetage-related macular degeneration. Cimzia (certolizumab pegol), is anAnti-TNF-α, PEGylated humanized fAb developed by UCB (Nelson andReichert, 2009) that is used to prevent moderate to severe Crohn'sdisease.

In certain embodiments, PET (Positron Emission Tomography) tracers areused as imaging agents. In certain embodiments, PET tracers comprise⁸⁹Zr, ⁶⁴Cu, [¹⁸F] fluorodeoxyglucose.

In certain embodiments, fluorophores comprise fluorochromes,fluorochrome quencher molecules, any organic or inorganic dyes, metalchelates, or any fluorescent enzyme substrates, including proteaseactivatable enzyme substrates. In certain embodiments, fluorophorescomprise long chain carbophilic cyanines. In other embodiments,fluorophores comprise DiI, DiR, DiD, and the like. Fluorochromescomprise far red, and near infrared fluorochromes (NIRF). Fluorochromesinclude but are not limited to a carbocyanine and indocyaninefluorochromes. In certain embodiments, imaging agents comprisecommercially available fluorochromes including, but not limited toCy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680,AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680,and VivoTag-5750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780(Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyteFluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, andIRDye 700DX (Li-Cor); and ADS780WS, ADS830WS, and ADS832WS (American DyeSource) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751(Carestream Health).

In certain embodiments, click reactive groups are used (for ‘clickchemistry’). Examples of click reactive groups include the following:alkyne, azide, thiol (sulfydryl), alkene, acrylate, oxime, maliemide,NHS (N-hydroxysuccinimide), amine (primary amine, secondary amine,tertiary amine, and/or quarternary ammonium), phenyl, benzyl, hydroxyl,carbonyl, aldehyde, carbonate, carboxylate, carboxyl, ester, methoxy,hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal,orthoester, orthocarbonate ester, amide, carboxyamide, imine (primaryketimine, secondary ketamine, primary aldimine, secondary aldimine),imide, azo (diimide), cyanate (cyanate or isocyanate), nitrate, nitrile,isonitrile, nitrite (nitrosooxy group), nitro, nitroso, pyridyl,sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanate,isothiocyanate, caronothioyl, thione, thial, phosphine, phosphono,phosphate, phosphodiester, borono, boronate, bomino, borinate, halo,fluoro, chloro, bromo, and/or iodo moieties.

Cancers that may be treated include, for example, prostate cancer,breast cancer, testicular cancer, cervical cancer, lung cancer, coloncancer, bone cancer, glioma, glioblastoma, multiple myeloma, sarcoma,small cell carcinoma, melanoma, renal cancer, liver cancer, head andneck cancer, esophageal cancer, thyroid cancer, lymphoma, and/orleukemia.

In certain embodiments, targeting peptide ligands, such as alpha-MSH,attached to C dots, can serve as immunomodulators alongside othertherapies to enhance treatment response.

In certain embodiments, in addition to administration of animmunoconjugate described herein, a method of treatment may includeadministration of antibodies, small molecule drugs, radiation,pharmacotherapy, chemotherapy, cryotherapy, thermotherapy,electrotherapy, phototherapy, ultrasonic therapy and/or surgery.

In certain embodiments, the immunoconjugate comprises a therapeuticagent, e.g., a drug (e.g., a chemotherapy drug) and/or a therapeuticradioisotope. As used herein, “therapeutic agent” refers to any agentthat has a therapeutic effect and/or elicits a desired biological and/orpharmacological effect, when administered to a subject.

In certain embodiments, the radioisotope is a radiolabel that can bemonitored/imaged (e.g., via PET or single-photon emission computedtomography (SPECT)). Example radioisotopes that can be used include betaemitters (e,g. ¹⁷⁷Lutetium) and alpha emitters (e.g., ²²⁵Ac). In certainembodiments, one or more of the following radioisotopes are used:^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁷⁷Lu, ⁶⁷Cu, ¹²³I,¹²⁴I, ¹²⁵I, ¹¹C, ¹3N, ¹⁵O, ¹⁸F, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁴⁹Pm, ⁹⁰Y, ²¹³Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ⁸⁹Zr, ²²⁵Ac, and ¹⁹²Ir.

In certain embodiments, the immunoconjugate comprises one or more drugs,e.g., one or more chemotherapy drugs, such as sorafenib, paclitaxel,docetaxel, MEK162, etoposide, lapatinib, nilotinib, crizotinib,fulvestrant, vemurafenib, bexorotene, and/or camptotecin.

In certain embodiments, the immunoconjugate comprises a chelator, forexample, 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diaceticacid (CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid(DTPA); 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid(DOTA); thylenediaminetetraacetic acid (EDTA); ethyleneglycolbis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA);1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA);ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-Cl-EHPG; 5Br-EHPG;5-Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA;benzyl-DTPA; dibenzyl DTPA; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivativesthereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1,4,7-triazacyclononaneN,N′,N″-triacetic acid (NOTA); benzo-NOTA; benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA);triethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and1,3,5-N,N′,N″-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM), orother metal chelators.

In certain embodiments, the immunoconjugate comprises more than onechelator.

In certain embodiments the radioisotope-chelator pair is ⁸⁹Zr-DFO. Incertain embodiments the radioisotope-chelator pair ¹⁷⁷Lu-DOTA. Incertain embodiments, the is radioisotope-chelator pair is ²²⁵Ac-DOTA.

In certain embodiments, the therapeutic agent (e.g., drug and/orradioisotope) is attached to the nanoparticle or the antibody fragment(protein), or both, using a bioorthogonal conjugation approach (e.g.,amine/NHS-ester, thiol/maleimide, azide/alkyne click, or tetrazine/TCOclick). For radiolabeling using radiometals, the radiometal chelator canbe first attached to either particle or protein or both, followed by theradiometal. Alternatively, the radiometal/chelator complex can beperformed, followed by attachment onto the particle or protein or both.Radioiodination can also be achieved using standard approaches where atyrosine or phenolic group on the particle or protein or both ismodified by electrophilic addition chemistry.

In certain embodiments, the immunoconjugate is administered to a subjectsuffering from a particular disease or condition (e.g., cancer) fortreatment of the disease or condition.

EXPERIMENTAL EXAMPLES Preparation of the C Dot-(Cy5)-PEG-Maleimide:

A maleimide and NHS ester functionalized polyethylene glycol(mal-dPEG₁₂-NHS) was conjugated with aminosilane (APTES) in DMSO (molarratio mal-PEG-NHS:APTES:DMSO 1:0.9:60). The reaction mixture was leftunder nitrogen at room temperature for 48 hours to generate silanefunctionalized mal-dPEG (mal-dPEG-APTES). A maleimide functionalized Cy5(mal-Cy5) was reacted with a thiol-silane (MPTMS) in DMSO (molar ratioCy5:MPTMS:DMOS 1:25:1150). The reaction was left under nitrogen at roomtemperature for 24 hours to generate a silane functionalized Cy5(Cy5-MPTMS). TMOS and Cy5-MPTMS were then titrated into an ammoniahydroxide solution (˜pH 8) (molar ratio TMOS:Cy5:NH3:H2O1:0.001:0.44:1215). The solution was stirred at 600 rpm at roomtemperature for 24 hours to form homogeneous Cy5 encapsulated silicananoparticles. The mal-dPEG-APTES and silane functionalized polyethyleneglycol (PEG-silane, MW around 500, Gelest) were then added into thesynthesis solution to PEGylate and surface-functionalize the particles(PEG-silane:TMOS:mal-PEG-APTES 1:2.3:0.006). The solution was stirred at600 rpm at room temperature for 24 hours followed by incubation at 80°C. for another 24 hours without stirring. The solution was dialyzed in2000 mL with deionized water for two days (10 k MWCO), filtered with 200nm syringe filters, and finally chromatographically purified (Superdex200) resulting in the desired mal-C dots.

Preparation of C Dot Immunoconjugates

Studies were performed to conjugate single chain antibody fragments(scFv)s to the C dot core silica nanoparticles. An scFv that boundmatrix metalloproteinase 12 (MMP-12) was expressed in E. coli. Theconstruct contained C-terminal His and FLAG tags for nickel affinitychromatography and immune-detection. A mutant scFv was constructed inwhich the last amino acid of the polypeptide chain was converted to acysteine (Cys). The change was confirmed by sequencing the mutant gene.Expression and nickel affinity purification of the wild type scFv andthe C-terminal Cys containing mutant was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), visualized withCoomassie blue stain at a molecular weight consistent with the scFv.Western blot analysis of the scFv SDS PAGE gel was performed with ananti-FLAG tag HRP conjugate. The Western blot analysis confirmed thatthe identity of the gel band was the scFv.

The scFv were clones modified with azide containing bifunctionallinkers. The wild type scFv was modified with N-hydroxy-succinimide(NHS) ester-polyethylene glycol (PEG)₄-azide. Without wishing to bebound to any theory, modification of wild type scFv with NHSester-PEG₄-azide results in the random incorporation of PEG₄-azide on tofree amines on surface lysine residues. The C-terminal scFv Cysconstruct was conjugated with maleimide-PEG₃-azide for site specificPEG₃-azide introduction on to the Cys sulfhydryl. The scFv constructswere analyzed for azide incorporation by reaction with aDibenzocyclooctyne (DBCO)-PEG-Cy5 fluorescent probe. Azides react withDBCOs via a metal free click chemistry reaction to form a covalentlinkage. Unreacted DBCO-Cy5 dye was removed from the reaction mixturesby 40 kDa cutoff size exclusion spin columns. The successfulintroduction of an azide group on the surface of the scFvs was confirmedby visualizing the wild type and C-terminal Cys scFv-PEG-Cy5 fluorescentdye constructs using a BioRad Versa-Doc imager.

The azide conjugated scFv were then reacted with C dots containing 1-3DBCOs on their surfaces. The reaction was allowed to continue for 12 hat room temperature. Unconjugated scFv was purified from conjugatedscFv-C dots using multiple techniques including phosphate bufferedsaline washes in 50,000 molecular weight cut off spin columns, G-200size exclusion column chromatography or size exclusion spin columns andvelocity sedimentation thought a sucrose cushion. Velocity sedimentationand size exclusion chromatography appear to be the most scalable methodsof purification. The purified scFv C-dot conjugates were analyzed by dotblot scFv immune-detection/particle fluorescence assays, gelelectrophoresis and fluorescent ELISAs with immobilized MMP-12.

These methods can be applied to other types of antibody fragments, e.g.,sdAbs.

FIG. 1 shows a schematic illustration showing the synthesis of⁸⁹Zr-labeled C′dot radioimmunoconjugate using a chelator-basedradiolabeling technique. PEGylated and maleimide-functionalized C′ dot(C′ dot-PEG-Mal, 1) was first reacted with reduced glutathione (GSH) tointroduce the —NH₂ groups for the following-up bioconjugates, forming C′dot-PEG-GSH (2). Then the nanoparticle was conjugated with DBCO-PEG4-NHSester and DFO-NCS, forming C′ dot-PEG-DBCO (3) and DFO-C′ dot-PEG-DBCO(4), respectively. Azide-functionalized small targeting ligands, such assingle-chain variable fragment (scFv-azide) (or single-domain antibody,sdAb-azide), was conjugated to the nanoparticle based on strain-promotedazide-alkyne cycloaddition, forming DFO-C′ dot-PEG-scFv (5). The finalC′dot radioimmunoconjugate (⁸⁹Zr-DFO-C′ dot-PEG-scFv, 6) was by labelingit with ⁸⁹Zr-oxalate. The schematic illustrated in FIG. 1 is not limitedto scFv and can include various types of antibody fragments, e.g.,sdAbs.

FIGS. 2A and 2B show in vivo (FIG. 2A) coronal and (FIG. 2B) sagittalPET images of ⁸⁹Zr-DFO-C′ dot-PEG at different post-injection timepoints (10 min, 1 h, Day 1, Day 3 and Day 6) in a healthy nude mouse.The reaction ratio between C′ dot-PEG-Mal and GSH was kept at 1:20. ThePET images were acquired by using a Focus 120 MicroPET scanner.

FIG. 3 shows biodistribution data of ⁸⁹Zr-DFO-C′ dot-PEG in a healthynude mouse on Day 6. Less than 2% ID/g of bone (and joint) uptake wasobserved.

FIGS. 4A and 4B show a chelator-free ⁸⁹Zr radiolabeling experimentalexample.

FIG. 4A shows ⁸⁹Zr labeling yields of C′ dot-PEG-Mal under varied pHconditions at 75° C.

FIG. 4B shows ⁸⁹Zr labeling yields of C′ dot-PEG-Mal using variedcombinations of C′ dot to ⁸⁹Zr-oxalate ratio.

FIGS. 5A and 5B show in vivo coronal PET images of [89Zr]C′ dot-PEG atdifferent post-injection time points (10 min, Day 1, Day 3 and Day 6) ina healthy nude mouse. [⁸⁹Zr]C′ dot-PEG was synthesized by using achelator-free radiolabeling technique.

The PET images were acquired by using a Focus 120 MicroPET scanner.

FIG. 5A shows PET images acquired without EDTA(ethylenediaminetetraacetic acid).

FIG. 5B shows PET images acquired with EDTA

FIG. 6 shows biodistribution data of [⁸⁹Zr]C′ dot-PEG in healthy nudemice (n=3) on Day 7. Over 10% ID/g of bone (and joint) uptake(highlighted with a red box) was observed in this case, indicating aless stable radiolabeling using a chelator-free method (when comparedwith that of chelator-based method).

FIG. 7 shows biodistribution data of ⁸⁹Zr-DFO-C′ dot, ⁸⁹Zr-DFO-C′dot-DBCO and ⁸⁹Zr-DFO-C′ dot-PEG-sdAb in healthy nude mice at 48 hpost-injection. An improved pharmacokinetic profile (with prolongedblood circulation half-life and lower liver uptake) can be achieved byoptimizing the number of DFO, DBCO and sdAb from each C′ dot.

1-44. (canceled)
 45. An immunoconjugate comprising: a nanoparticlecoated with an organic polymer; an antibody fragment conjugated to theorganic polymer-coated nanoparticle, a therapeutic agent conjugated tothe organic polymer-coated nanoparticle through a linker, wherein thenanoparticle has a diameter no greater than 20 nanometers, wherein thenanoparticle comprises a silica-based core and a silica shellsurrounding at least a portion of the core, wherein the antibodyfragment is a single chain variable fragment (scFv).
 46. Theimmunoconjugate of claim 45, wherein the linker is a cleavable linker.47. The immunoconjugate of claim 46, wherein the cleavable linker isselected from a group consisting of peptide, hydrazine and disulfidelinkers.
 48. The immunoconjugate of claim 45, wherein the linker is apeptide linker.
 49. The immunoconjugate of claim 45, wherein the linkeris a peptide linker that is cleaved by lysosomal proteases.
 50. Theimmunoconjugate of claim 49, wherein the lysosomal protease iscathepsin-B.
 51. The immunoconjugate of claim 45, wherein the linker isa dipeptide linker.
 52. The immunoconjugate of claim 51, wherein thedipeptide linker is a valine-citrulline linker.
 53. The immunoconjugateof claim 45, wherein the antibody fragment is from about 25 kDa to about30 kDa.
 54. The immunoconjugate of claim 45, wherein the nanoparticlecomprises a fluorescent compound within the core.
 55. Theimmunoconjugate of claim 45, wherein the nanoparticle has from one toten antibody fragments attached thereto.
 56. The immunoconjugate ofclaim 45, wherein the nanoparticle has a diameter no greater than 15nanometers.
 57. The immunoconjugate of claim 45, wherein thenanoparticle has a diameter in a range from 1 nm to 20 nm.
 58. Theimmunoconjugate of claim 45, wherein the antibody fragment comprisesanti-VEGF-A.
 59. The immunoconjugate of claim 45, wherein theimmunoconjugate comprises one or more imaging agents.
 60. Theimmunoconjugate of claim 59, wherein the one or more imaging agentscomprise a PET tracer.
 61. The immunoconjugate of claim 59, wherein theone or more imaging agents comprise a fluorophore.
 62. Theimmunoconjugate of claim 4, wherein the therapeutic agent comprises achemotherapy drug.
 63. The immunoconjugate of claim 45, wherein thetherapeutic agent comprises a radioisotope.
 64. The immunoconjugate ofclaim 63, wherein the radioisotope is a member selected from the groupconsisting of ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁷⁷Lu,⁶⁷Cu, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N, ¹⁵O, ¹⁸F, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹³Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ⁸⁹Zr, ²²⁵Ac, and ¹⁹²Ir.